High affinity humanized anti-Tag-72 monoclonal antibodies

ABSTRACT

Novel humanized monoclonal antibodies, humanized antibody fragments, and derivatives thereof which specifically bind TAG-72 are provided as well as methods for their manufacture. These humanized antibodies are useful in the treatment of cancers which express TAG-72 as well as for diagnostic purposes, e.g., for in vivo imaging of tumors or cancer cells which express TAG-72.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present Application is a Division of U.S. patent Application Ser.No. 09/025,203, filed Feb. 18, 1998, now U.S. Pat. No. 6,348,581, whichis a Continuation-in-Part of International Application Serial No.PCT/US97/19641, filed Oct. 30, 1997 designating the United States, nowabandoned, which is a Continuation-in-Part of U.S. Provisional PatentApplication Serial No. 60/030,173, filed Oct. 31, 1996, now abandoned.

FIELD OF THE INVENTION

The present invention relates to humanized monoclonal antibodies andfragments or derivatives thereof which specifically bindtumor-associated glycoprotein TAG-72, a human pancarcinoma antigenexpressed by various human tumor cells. More specifically, the presentinvention relates to humanized monoclonal antibodies and fragments orderivatives thereof derived from murine monoclonal antibody CC49 orother murine antibodies which specifically bind TAG-72. The presentinvention further relates to methods for producing such humanizedmonoclonal antibodies specific to TAG-72, pharmaceutical and diagnosticcompositions containing such humanized monoclonal antibodies, andmethods of use thereof for the treatment or diagnosis of cancer.

BACKGROUND OF THE INVENTION

The identification of antigens expressed by tumor cells and thepreparation of monoclonal antibodies which specifically bind suchantigens is well known in the art. Anti-tumor monoclonal antibodiesexhibit potential application as both therapeutic and diagnostic agents.Such monoclonal antibodies have potential application as diagnosticagents because they specifically bind tumor antigens and thereby candetect the presence of tumor cells or tumor antigen in an analyte. Forexample, use of monoclonal antibodies which bind tumor antigens for invitro and in vivo imaging of tumor cells or tumors using a labeled formof such a monoclonal antibody is conventional in the art.

Moreover, monoclonal antibodies which bind tumor antigens have wellknown application as therapeutic agents. The usage of monoclonalantibodies themselves as therapeutic agents, or as conjugates whereinthe monoclonal antibody is directly or indirectly attached to aneffector moiety, e.g., a drug, cytokine, cytotoxin, etc., is well known.

Essentially, if the monoclonal antibody is attached to an effectormoiety the monoclonal antibody functions as a targeting moiety, i.e. itdirects the desired effector moiety (which typically possessestherapeutic activity) against a desired target, e.g., a tumor whichexpresses the antigen bound by the monoclonal antibody. In contrast,when the monoclonal antibody itself operates as a therapeutic agent, theantibody functions both as a targeting moiety—i.e., it will specificallybind a cell which expresses the antigen—and as an effector whichmediates therapeutic activity, typically tumor cell Iysis. Such effectorfunctions—including, e.g., antibody-dependent cellular cytotoxicity(ADCC) and complement dependent cytotoxicity (CDC), among others—areeffected by the portion of the antibody molecule generally referred toin the literature as the Fc portion. One specific tumor antigen againstwhich various monoclonal antibodies have been developed istumor-associated glycoprotein TAG-72. TAG-72 is expressed on the surfaceof various human tumor cells, such as the LS174T tumor cell line(American Type Tissue Collection (ATCC) No. CL188, a variant of cellline LS180 (ATCC No. CL187)), a colon adenocarcinoma line. Variousresearch groups have reported the production of monoclonal antibodies toTAG-72. See, e.g., Muraro et al., Cancer Res., 48:4588-4596 (1988);Johnson et al., Cancer Res., 46:850-857 (1986); Molinolo et al., CancerRes., 50:1291-1298 (1990); Thor et al., Cancer Res., 46:3118-3127(1986); EP 394,277 to Schlom et al. (assigned to the National CancerInstitute); and U.S. Pat. No. 5,512,443 to Jeffrey Schlom et al.Specific antibodies to TAG-72 which are publicly available include CC49(ATCC No. HB 9459), CC83 (ATCC No. HB 9453), CC46 (ATCC No. HB 9458),CC92 (ATCC No. HB 9454), CC30 (ATCC No. HB 9457), CC11 (ATCC No. 9455),and CC15 (ATCC No. HB 9460).

One example thereof, CC49, is a murine monoclonal antibody of the IgG₁isotype. This monoclonal antibody is a second generation monoclonalantibody prepared by immunizing mice with TAG-72 purified using thefirst generation antibody B72.3. Colcher et al., Proc. Natl. Acad. Sci.USA, 78:3199-3203 (1981). CC49 specifically binds TAG-72, and has ahigher antigen-binding affinity than B72.3. Muraro et al., Cancer Res.,48:4588-4596 (1988). This monoclonal antibody has been reported totarget human colon carcinoma xenografts efficiently, and to reduce thegrowth of such xenografts with good efficacy. Molinolo et al., CancerRes., 50:1291-1298 (1996); Colcher et al., J. Natl. Cancer Inst.,82:1191-1197 (1990). Also, radiolabeled CC49 has been reported toexhibit excellent tumor localization in several ongoing clinical trials.

However, while murine antibodies have applicability as therapeuticagents in humans, they are disadvantageous in some respects.Specifically, murine antibodies, because of the fact that they are offoreign species origin, may be immunogenic in humans. This may result ina neutralizing antibody response (human anti-murine antibody (HAMA)response), which is particularly problematic if the antibodies aredesired to be administered repeatedly, e.g., in treatment of a chronicor recurrent disease condition. Also, because they contain murineconstant domains they may not exhibit human effector functions.

In an effort to eliminate or reduce such problems, chimeric antibodieshave been disclosed. Chimeric antibodies contain portions of twodifferent antibodies, typically of two different species. Generally,such antibodies contain human constant regions and variable regions ofanother species, typically murine variable regions. For example, somemouse/human chimeric antibodies have been reported which exhibit bindingcharacteristics of the parental mouse antibody, and effector functionsassociated with the human constant region. See, e.g.: U.S. Pat. No.4,816,567 to Cabilly et al.; U.S. Pat. No. 4,978,745 to Shoemaker etal.; U.S. Pat. No. 4,975,369 to Beavers et al.; and U.S. Pat. No.4,816,397 to Boss et al. Generally, these chimeric antibodies areconstructed by preparing a genomic gene library from DNA extracted frompre-existing murine hybridomas. Nishimura et al., Cancer Res., 47:999(1987). The library is then screened for variable region genes from bothheavy and light chains exhibiting the correct antibody fragmentrearrangement patterns. Alternatively, cDNA libraries are prepared fromRNA extracted from the hybridomas and screened, or the variable regionsare obtained by polymerase chain reaction. The cloned variable regiongenes are then ligated into an expression vector containing clonedcassettes of the appropriate heavy or light chain human constant regiongene. The chimeric genes are then expressed in a cell line of choice,usually a murine myeloma line. Such chimeric antibodies have been usedin human therapy.

Moreover, the production of chimeric mouse-human antibodies derived fromCC49 and CC83, which specifically bind TAG-72, has been reported. Inthis regard, see e.g., EPO 0,365,997 to Mezes et al. (The Dow ChemicalCompany). One such chimeric CC49 antibody is that produced by the cellline deposited as ATTC No. HB 9884 (Budapest).

Also, Morrison et al. report the preparation of several antitumorchimeric monoclonal antibodies, in Important Advances in Oncology,Recombinant Chimeric Monoclonal Antibodies, pp. 3-18 (S. A. Rosenberg,ed., 1990) (J. B. Lippincott, Philadelphia, Pa.). Results of clinicaltrials with chimeric cMAb-17-1A in patients with metastatic colorectalcarcinoma now show that this antibody has a 6-fold longer circulationtime and significantly reduced immunogenicity as compared to the murinemonoclonal antibody from which it was derived. LoBuglio et al., Proc.Natl. Acad. Sci. USA, 86:4220-4224 (1989); Meredith et al., J. Nucl.Med., 32:1162-1168 (1991).

However, while such chimeric monoclonal antibodies typically exhibitlesser immunogenicity, they are still potentially immunogenic in humansbecause they contain murine variable sequences which may elicit antibodyresponses. Thus, there is the possibility that these chimeric antibodiesmay elicit an anti-idiotypic response if administered to patients. Salehet al., Cancer Immunol. Immunother., 32:185-190 (1990).

For example, when cB72.3(γ4) was administered to patients withcolorectal carcinomas, 62% of such patients elicited a human antimurineantibody (HAMA) response, which included an anti-V-region response. Thisis disadvantageous because a HAMA response would make repeated antitumorantibody administration potentially ineffective because of an increasedantibody clearance from the serum (Saleh et al., Cancer Immunol.Immunother., 32:180-190 (1990)) and also because of potential allergicreactions (LoBuglio et al., Hybridoma, 5:5117-5123 (1986).

A number of genetic variants of potential clinical utility have beendeveloped from MAb CC49. These include cCC49, a C_(H)2 domain-deficientcCC49 (Slavin-Chiorini et al, Int. J. Cancer, 53:97-103 (1993)), and asingle chain Fv (sFv) (Milenic et al., Cancer Res., 51:6365-6371 (1991);Sawyer et al., Protein Eng., 7:1401-1406 (1994)). These molecules mayelicit relatively reduced HAMA responses in patients, since they haveshown more rapid plasma and whole body clearance rates in mice andrhesus monkeys, as compared to intact IgG. Slavin-Chiorini et al. (1993)(id.); Milenic et al. (1991) (id.). Additionally, novel single-chainimmunoglobulin (SC1 g) molecules derived from cCC49 have been reportedand are encoded by single-gene constructs. One such molecule,SCIgΔC_(H)1 consists of CC49 sFv linked to the human γ1 Fc region (Shuet al., Proc. Natl. Acad. Sci. USA, 90:7995-7999 (1993)) while the otherSCIg-IL-2 carries a human interleukin-2 (IL-2) molecule geneticallyattached to the carboxyl end of the Fc region of SCIgΔC_(H)1 (Kashmiriet al., Proc. XVI Intl. Cancer Cong., 1:183-187 (1994)). Both SCIgs arecomparable to cCC49 in antigen binding and antibody cellular cytolyticactivity. The biological activity of the IL-2 is also retained inSCIg-IL-2.

In an effort to alleviate the immunogenicity concerns of chimeric andmurine antibodies, the production of “humanized” antibodies is alsoknown. Ideally, “humanization” results in an antibody that isnon-immunogenic in humans, with substantially complete retention of theantigen-binding properties of the original molecule. In order to retainall the antigen-binding properties of the original antibody, thestructure of its combining-site has to be faithfully reproduced in the“humanized” version. This can potentially be achieved by transplantingthe combining site of the nonhuman antibody onto a human framework,either (a) by grafting only the nonhuman CDRs onto human framework andconstant regions with or without retention of critical frameworkresidues (Jones et al., Nature, 321:522 (1986); Verhoeyen et al.,Science, 239:1539 (1988)); or (b) by transplanting the entire nonhumanvariable domains (to preserve ligand-binding properties) but also“cloaking” them with a human-like surface through judicious replacementof exposed residues (to reduce antigenicity) (Padlan, Molec. Immunol.,28:489 (1991)).

Essentially, humanization by CDR grafting involves transplanting onlythe CDRs onto human fragment and constant regions. Theoretically, thisshould substantially eliminate immunogenicity (except if allotypic oridiotypic differences exist). Jones et al., Nature, 321:522-525 (1986);Verhoeyen et al., Science, 239:1534-1536 (1988); Riechmann et al.,Nature, 332:323-327 (1988). While such a technique is effective in someinstances, CDR-grafting sometimes does not yield the desired result.Rather, it has been reported that some framework residues of theoriginal antibody may also need to be preserved in order to preserveantigen binding activity. Riechmann et al., Nature, 332:323-327 (1988);Queen et al., Proc. Natl. Acad. Sci. USA, 86:10023-10029; Tempest etal., Biol. Technology, 9:266-271 (1991); Co et al., Nature, 351:501-502(1991)).

As discussed, in order to preserve the antigen-binding properties of theoriginal antibody, the structure of its combining site must befaithfully reproduced in the humanized molecule. X-ray crystallographicstudies have shown that the antibody combining site is built primarilyfrom CDR residues, although some neighboring framework residues havebeen found to be involved in antigen binding. Amit et al., Science,233:747-753 986); Colman et al., Nature, 326:358-363 (1987); Sheriff etal., Proc. Natl. Acad. Sci. USA, 84:8075-8079 (1987); Padlan et al.,Proc. Natl. Acad Sci. USA, 86:5938-5942 (1989); Fischmann et al., J.Biol. Chem., 266:12915-12920 (1991); Tulip et al., J. Molec. Biol.,227:122-148 (1992). It has also been found that the structures of theCDR loops are significantly influenced by surrounding frameworkstructures. Chothia et al., J. Molec. Biol., 196:901-917 (1987); Chothiaet al., Nature, 342:877-883 (1989); Tramomonteno et al., J. Molec. Biol,215:175-182 (1990).

Small but significant differences in the relative disposition of thevariable light chain (V_(L)) and variable heavy (V_(H)) domains havebeen noted (Colman et al., Nature, 326:358-363 (1987)) and thosedifferences are ostensibly due to variations in the residues involved inthe interdomain contact (Padlan et al., Molec. Immunol., 31:169-217(1994)).

Furthermore, structural studies of the effect of the mutation ofinterior residues, in which changes in side chain volume are involved,have shown that the resulting local deformations are accommodated byshifts in side chain positions that are propagated to distant parts ofthe molecular interior. This suggests that during humanization theinterior residues in the variable domains and in the interface betweenthese domains, or at least the interior volumes, should also bemaintained; a humanization protocol in which an interior residue isreplaced by one of different physical properties (such as size, charge,or hydrophobicity, etc.), could result in a significant modification ofthe antigen combining site structure.

One method of identifying the framework residues which need to bepreserved is by computer modeling. Alternatively, critical frameworkresidues may potentially be identified by comparing known antibodycombining site structures (Padlan, Molec. Immun., 31(3):169-217 (1994)).

The residues which potentially affect antigen binding fall into severalgroups. The first group comprises residues that are contiguous with thecombining site surface and which could therefore make direct contactwith antigens. They include the amino-terminal residues and thoseadjacent to the CDRs. The second group includes residues that couldalter the structure or relative alignment of the CDRs either bycontacting the CDRs or the opposite chains. The third group comprisesamino acids with buried side chains that could influence the structuralintegrity of the variable domains. The residues in these groups areusually found in the same positions (ibid.) according to the adoptednumbering system. See Kabat et al., Sequences of Proteins ofImmunological Interest, NIH Pub. No. 91-3242 (5th ed., 1991) (U.S. Dept.Health & Human Services, Bethesda, Md.) and Genbank.

However, while humanized antibodies are desirable because of theirpotential low immunogenicity in humans, their production isunpredictable. For example, sequence modification of antibodies mayresult in substantial or even total loss of antigen binding affinity, orloss of binding specificity. Alternatively, “humanized antibodies” maystill exhibit immunogenicity in humans, irrespective of sequencemodification.

Thus, there still exists a significant need in the art for novelhumanized antibodies to desired antigens. More specifically, thereexists a need in the art for humanized antibodies specific to TAG-72,because of their potential as immunotherapeutic and immunodiagnosticagents.

OBJECTS OF THE INVENTION

Toward this end, it is an object of the invention to provide humanizedantibodies which are specific to human TAG-72.

More specifically, it is an object of the invention to provide humanizedantibodies derived from murine antibodies to TAG-72, and in particularfrom CC49, a specific murine antibody which binds to TAG-72.

It is also an object of the invention to provide pharmaceuticalcompositions containing humanized antibodies which are specific toTAG-72. It is a more specific object of the invention to providepharmaceutical compositions containing humanized antibodies derived fromCC49, a murine antibody which specifically binds to TAG-72.

It is another specific object of the invention to provide methods ofusing humanized antibodies to TAG-72 for treatment of cancers whichexpress TAG-72, in particular human colon cancer.

It is another object of the invention to provide immunodiagnosticcompositions for detecting cancer cells which contain a humanizedantibody which specifically binds TAG-72, and preferably is derived fromCC49, which antibody is in labeled or unlabeled form. It is anotherobject of the invention to provide a method of immunodiagnosis of cancerusing compositions which contain a humanized antibody which specificallybinds TAG-72, which is in labeled or unlabeled form.

It is still another object of the invention to provide nucleic acidsequences which encode for humanized antibodies to TAG-72 or fragmentsthereof. It is a more specific object of the invention to providenucleic acid sequences which encode humanized antibodies derived fromCC49, a murine antibody which specifically binds to TAG-72. It isanother object of the invention to provide vectors from which may beexpressed humanized antibodies to TAG-72, in particular humanizedantibodies derived from CC49, a murine antibody which specifically bindsto TAG-72.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 aligns amino acid sequences of murine CC49 V_(H) (SEQ ID NO:1)the NEWM framework regions (SEQ ID NO:2) encoded by the FR startingmaterial, and the humanized NEWM-based V_(H) (HuVH) (SEQ ID NO:3)disclosed in Example 1. The CDRs are in the boxes. Murine residuesretained in the FRs are identified with arrow symbols (↑). Murine FRresidues retained in alternate versions of the HuVH (SEQ ID NOS: 4, 5,AND 6) are identified with letter symbols (A), (S), and (K).

FIG. 2 aligns amino acid sequences of murine CC49 V_(K) (SEQ ID NO:7),the REI framework regions (SEQ ID NO:8) encoded by the FR startingmaterial, and the humanized REI-based V_(K) (SEQ ID NO:9) disclosed inExample 1. CDRs are in the boxes.

FIG. 3 aligns the variable heavy chain of CC49 (SEQ ID NO:10), theHuCC49 (SEQ ID NO:11) disclosed in Example 1, and NEWM (SEQ ID NO:12).

FIG. 4 aligns the variable light chain of CC49 (SEQ ID NO:13), theHuCC49 (SEQ ID NO:14) disclosed in Example 1, and REI (SEQ ID NO:15).

FIG. 5 contains schematics of the vectors used to express the humanizedV_(H) and V_(K) shown in FIG. 3 and FIG. 4.

FIG. 6 is an ELISA showing binding of CC49 antibodies HuVHA/MuVK andHuVHA/HuVK to TAG-72.

FIG. 7 is an ELISA showing binding of CC49 antibodies MuVH/MuVK andHuVH/HuVK to TAG-72.

FIG. 8 is an ELISA showing binding of CC49 antibodies MuVH/MuVK andHuVH/HuVK to TAG-72.

FIG. 9 is an ELISA showing binding of CC49 antibodies MuVH/MuVK andHuVHA/HuVK to TAG-72.

FIG. 10 is an ELISA showing binding of CC49 antibodies HuVH/HuVK andHuVHK/HuVK to TAG-72.

FIG. 10 is an ELISA showing binding of CC49 antibodies HuVHS/HuVK andHuVH/HuVK to TAG-72.

FIG. 12 is a Scatchard analysis of humanized (HuVH/HuVK) and chimeric(MuVH/MuVK) CC49 monoclonal antibodies.

FIG. 13 presents the single-stranded DNA sequence (SEQ ID NO:16) of thetemplate used to produce the initial humanized NEWM-based VHs, HuVH andHuVHA.

FIG. 14 presents the double-stranded DNA sequence (SEQ ID NO:17) of thetemplate used to produce the alternate humanized VHs, HuVHS and HuVHK.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, definitions of certain terms whichare used in this disclosure are set forth below:

Antibody—This refers to single chain, two-chain, and multi-chainproteins and glycoproteins belonging to the classes of polyclonal,monoclonal, chimeric, and hetero immunoglobulins (monoclonal antibodiesbeing preferred); it also includes synthetic and genetically engineeredvariants of these immunoglobulins. “Antibody fragment” includes Fab,Fab′, F(ab′)₂, and Fv fragments, as well as any portion of an antibodyhaving specificity toward a desired target epitope or epitopes.

Humanized antibody—This will refer to an antibody derived from anon-human antibody, typically murine, that retains or substantiallyretains the antigen-binding properties of the parent antibody but whichis less immunogenic in humans. This may be achieved by various methodsincluding (a) grafting only the non-human CDRs onto human framework andconstant regions with or without retention of critical frameworkresidues, or (b) transplanting the entire non-human variable domains,but “cloaking” them with a human-like section by replacement of surfaceresidues. Such methods as are useful in practicing the present inventioninclude those disclosed in Jones et al., Morrison et al., Proc. Natl.Acad. Sci. USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol.,44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988);Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun.,31(3):169-217 (1994).

Complementarity Determining Region. or CDR—The term CDR, as used herein,refers to amino acid sequences which together define the bindingaffinity and specificity of the natural Fv region of a nativeimmunoglobulin binding site as delineated by Kabat et al. (1991).

Framework Region—The term FR, as used herein, refers to amino acidsequences interposed between CDRs. These portions of the antibody serveto hold the CDRs in an appropriate orientation for antigen binding. Inthe antibodies and antibody fragments of the present invention, theframework regions for the light chain variable region may be selectedfrom the group consisting of human lambda light chain FRs and humankappa subgroup I, II, and III light chain FRs, whether comprising theirfully human native amino acid sequences or comprising amino acidsequence modifications necessary to retain or increase binding affinityand/or binding specificity.

Constant Region—The portion of the antibody molecule which conferseffector functions. In the present invention, murine constant regionsare substituted with human constant regions. The constant regions of thesubject chimeric or humanized antibodies are derived from humanimmunoglobulins. The heavy chain constant region can be selected fromany of the five isotypes: alpha, delta, epsilon, gamma or mu. Further,heavy chains of various subclasses (such as the IgG subclasses of heavychains) are responsible for different effector functions and thus, bychoosing the desired heavy chain constant region chimeric antibodieswith desired effector function can be produced.

Preferred constant regions are gamma 1 (IgG1), gamma 3 (IgG3) and gamma4 (IgG4). More preferred is a constant region of the gamma 1 (IgG1)isotype. The light chain constant region can be of the kappa or lambdatype, preferably of the kappa type.

Chimeric antibody—This is an antibody containing sequences derived fromtwo different antibodies, which typically are of different species. Mosttypically chimeric antibodies comprise human and murine antibodyfragments, generally human constant and murine variable regions.

Mammals—Animals that nourish their young with milk secreted by mammaryglands, preferably warm blooded mammals, more preferably humans.

Immunogenicity—A measure of the ability of a targeting protein ortherapeutic moiety to elicit an immune response (humoral or cellular)when administered to a recipient. The present invention is concernedwith the immunogenicity of the subject humanized antibodies or fragmentsthereof.

Humanized antibody of reduced immunogenicity—This refers to a humanizedantibody exhibiting reduced immunogenicity relative to the parentantibody.

Humanized antibody substantially retaining the binding properties of theparent antibody—This refers to a humanized antibody which retains theability to specifically bind the antigen recognized by the parentantibody used to produce such humanized antibodies. Preferably thehumanized antibody will exhibit the same or substantially the sameantigen-binding affinity and avidity as the parent antibody, e.g., CC49.Preferably, the affinity of the antibody will at least about 10% of thatof the parent antibody. More preferably, the affinity will be at leastabout 25%, i.e. at least two-fold less than the affinity of the parentantibody. Most preferably the affinity will be at least about 50% thatof the parent antibody. Methods for assaying antigen-binding affinityare well known in the art and include half-maximal binding assays,competition assays, and Scatchard analysis. Suitable antigen bindingassays are described in this application.

In its broadest embodiment, the present invention is directed tohumanized antibodies which specifically bind TAG-72, a pancarcinomaantigen expressed by various human cancers, in particular human coloncarcinoma. Preferably, such humanized antibodies will be derived fromantibodies having good binding affinity to TAG-72, e.g.: B72.3 (Thor etal., Cancer Res., 46:3118-3127 (1986); Johnson et al., Cancer Res.,46:850-857 (1986)), deposited as ATCC No. HB 8108; CC49 (ATCC No. HB9459); CC83 (ATCC No. HB 9453); CC46 (ATCC No. HB 9452); CC92 (ATCC No.HB 9454); CC30 (ATCC No. HB 9457); CC11 (ATCC No. 9455); and CC15 (ATCCNo. HB 9460); or chimerized forms thereof (see, e.g., EPO 0,365,997 toMezes et al., The Dow Chemical Company).

Most preferably, such humanized antibodies will be derived from CC49,which has been reported to target human colon carcinoma xenograftsefficiently and also to reduce the growth of the xenograft with goodefficacy. Molinolo et al., Cancer Res., 50:1291-1298 (1990); Colcher etal., J. Natl. Cancer Inst., 82:1191-1197 (1990).

As discussed above, humanized antibodies afford potential advantagesover murine and also chimeric antibodies, e.g., reduced immunogenicityin humans. This is advantageous because it should reduce and potentiallyeliminate the eliciting of a HAMA response when such humanizedantibodies are administered in vivo, e.g., for treatment of cancer orfor diagnosis of cancer, e.g., for tumor imaging.

However, as noted, humanization may in some instances adversely affectantigen binding. Preferably, the humanized antibodies of the presentinvention which specifically bind TAG-72 will possess a binding affinityfor TAG-72 of at least about 10% and more preferably at least about 25%and most preferably at least about 50% that of the TAG-72 antigenbinding affinity of the parent murine antibody, e.g., B72.3, CC49, CC46,CC30, CC11, CC15, CC83, or another parent antibody. Most preferably, thehumanized antibodies of the present invention will possess a bindingaffinity for TAG-72 of at least about 10% and more preferably at leastabout 25% and most preferably at least about 50% that of the TAG-72antigen binding affinity of either CC49 or a chimeric CC49 antibody.

Preferably, the humanized antibodies of the present invention will bindthe same epitope as CC49. Such antibodies can be identified based ontheir ability to compete with CC49 for binding to TAG-72 or toTAG-72-expressing cancer cells.

In general, the subject humanized antibodies are produced by obtainingnucleic acid sequences encoding the variable heavy and variable lightsequences of an antibody which binds TAG-72, preferably CC49,identifying the CDRs in said variable heavy and variable lightsequences, and grafting such CDR nucleic acid sequences onto humanframework nucleic acid sequences.

Preferably, the selected human framework will be one that is expected tobe suitable for in vivo administration, i.e., does not exhibitimmunogenicity. This can be determined, e.g., by prior experience within vivo usage of such antibodies and by studies of amino acid sequencesimilarities. In the latter approach, the amino acid sequences of theframework regions of the antibody to be humanized, e.g., CC49, will becompared to those of known human framework regions, and human frameworkregions used for CDR grafting will be selected which comprise a size andsequence most similar to that of the parent antibody, e.g., a murineantibody which binds TAG-72. Numerous human framework regions have beenisolated and their sequences reported in the literature. See, e.g.,Kabat et al., (id.).This enhances the likelihood that the resultantCDR-grafted “humanized” antibody, which contains the CDRs of the parent(e.g., murine) antibody grafted onto the selected human frameworkregions will significantly retain the antigen binding structure and thusthe binding affinity of the parent antibody. As a result of suchstudies, the FRs of REI and NEWM antibodies have been identified ashaving amino acid sequences which are likely to allow the CDRs of CC49to retain a significant degree of antigen binding affinity. As noted,the selected human framework regions will preferably be those that areexpected to be suitable for in vivo administration, i.e., notimmunogenic. Based on their amino acid sequences, REI and NEWM humanframework regions are expected to be substantially non-immunogenic.

Methods for cloning nucleic acid sequences encoding immunoglobulins arewell known in the art. Such methods will generally involve theamplification of the immunoglobulin sequences to be cloned usingappropriate primers by polymerase chain reaction (PCR). Primers suitablefor amplifying immunoglobulin nucleic acid sequences, and specificallymurine variable heavy and variable light sequences have been reported inthe literature. After such immunoglobulin sequences have been cloned,they will be sequenced by methods well known in the art. This will beeffected in order to identify the variable heavy and variable lightsequences, and more specifically the portions thereof which constitutethe CDRs and FRs. This can be effected by well known methods.

Once the CDRs and FRs of the cloned antibody sequences which are to behumanized have been identified, the amino acid sequences encoding CDRsare then identified (deduced based on the nucleic acid sequences and thegenetic code and by comparison to previous antibody sequences) and thecorresponding nucleic acid sequences are grafted onto selected humanFRs. This may be accomplished by use of appropriate primers and linkers.Methods for selecting suitable primers and linkers to provide forligation of desired nucleic acid sequences is well within the purview ofthe ordinary artisan and include those disclosed in U.S. Pat. No.4,816,397 to Boss et al. and U.S. Pat. No. 5,225,539 to Winter et al.

After the CDRs are grafted onto selected human FRs, the resultant“humanized” variable heavy and variable light sequences will then beexpressed to produce a humanized Fv or humanized antibody which bindsTAG-72. Typically, the humanized variable heavy and variable lightsequences will be expressed as a fusion protein with human constantdomain sequences, so that an intact antibody which binds TAG-72 isobtained. However, this is not necessary as the variable heavy and lightsequences can also be expressed in the absence of constant sequences toproduce a humanized Fv which binds TAG-72. However, fusion of humanconstant sequences to the humanized variable region(s) is potentiallydesirable because the resultant humanized antibody which binds TAG-72will then possess human effector functions such as complement-dependentcytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)activity. Such activity has been found in chimeric antibodies, includingCC49. The humanized anti-TAG-72 antibodies of the present invention canalso support such effector function activity with the added advantage ofa greatly decreased risk of a HAMA response.

Methods for synthesizing DNAs encoding a protein of known sequence arewell known in the art. Using such methods, DNA sequences which encodethe subject humanized V_(L) and V_(H) sequences are synthesized, andthen expressed in vector systems suitable for expression of recombinantantibodies. This may be effected in any vector system which provides forthe subject humanized V_(L) and V_(H) sequences to be expressed as afusion protein with human constant domain sequences and to associate toproduce functional (antigen binding) antibodies. Expression vectors andhost cells suitable for expression of recombinant antibodies andhumanized antibodies in particular, are well known in the art.

The following references are representative of methods and vectorssuitable for expression of recombinant immunoglobulins which may beutilized in carrying out the present invention. Weidle et al., Gene,51:21-29 (1987); Dorai et al., J. Immunol., 13(12):4232-4241 (1987); DeWaele et al., Eur. J. Biochem., 176:287-295 (1988); Colcher et al.,Cancer Res., 49:1738-1745 (1989); Wood et al., J. Immunol.,145(a):3011-3016 (1990); Bulens et al., Eur. J. Biochem., 195:235-242(1991); Beggington et al., Biol. Technology, 10:169 (1992); King et al.,Biochem. J., 281:317-323 (1992); Page et al., Biol. Technology, 9:64(1991); King et al., Biochem. J., 290:723-729 (1993); Chaudary et al.,Nature, 339:394-397 (1989); Jones et al., Nature, 321:522-525 (1986);Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Benhar et al., Proc.Natl. Acad. Sci. USA, 91:12051-12055 (1994); Singer et al., J. Immunol.,150:2844-2857 (1993); Cooto et al., Hybridoma, 13(3):215-219 (1994);Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989); Caronet al., Cancer Res., 32:6761-6767 (1992); Cotoma et al., J. Immunol.Meth., 152:89-109 (1992). Moreover, vectors suitable for expression ofrecombinant antibodies are commercially available. The vector may, e.g.,be a bare nucleic acid segment, a carrier-associated nucleic acidsegment, a nucleoprotein, a plasmid, a virus, a viroid, or atransposable element.

Host cells known to be capable of expressing functional immunoglobulinsinclude, e.g.: mammalian cells such as Chinese Hamster Ovary (CHO)cells; COS cells; myeloma cells, such as NSO and SP2/0 cells; bacteriasuch as Escherichia coli; yeast cells such as Saccharomyces cerevisiae;and other host cells. Of these, CHO cells are used by many researchersgiven their ability to effectively express and secrete immunoglobulins.NSO cells are one of the preferred types of host cells useful in thepresent invention.

Essentially, recombinant expression of humanized antibodies is effectedby one of two general methods. In the first method, the host cells aretransfected with a single vector which provides for the expression ofboth heavy and light variable sequences optionally fused to selectedconstant regions. In the second method, host cells are transfected withtwo vectors, each of which encodes a different variable chain (i.e. avariable heavy chain or variable light chain); each variablechain-encoding vector may optionally provide for the expression of thevariable chain fused to a selected constant region.

Human constant domain sequences are well known in the art, and have beenreported in the literature. Preferred human light chain constantsequences include the kappa and lambda constant light sequences.Preferred human heavy constant sequences include human gamma 1, humangamma 2, human gamma 3, human gamma 4, and mutated versions thereofwhich provide for altered effect or function, e.g., enhanced in vivohalf-life or reduced Fc receptor binding.

After expression, the antigen binding affinity of the resultinghumanized antibody will be assayed by known methods, e.g., Scatchardanalysis. In a particularly preferred embodiment, the antigen-bindingaffinity of the humanized antibody will be at least 25% of that of theparent antibody, e.g., CC49, i.e. a minimum of two-fold less than thatof native or chimeric CC49. Most preferably, the affinity of thehumanized antibody will be at least about 50% of that of the parentantibody, e.g., CC49.

In some instances, humanized antibodies produced by grafting CDRs (froman antibody which binds TAG-72) onto selected human framework regionsmay provide humanized antibodies having the desired affinity to TAG-72.However, it may be necessary or desirable to further modify specificresidues of the selected human framework in order to enhance antigenbinding. This may occur because it is believed that some frameworkresidues are essential to or at least affect antigen binding.Preferably, those framework residues of the parent (e.g., murine)antibody which maintain or affect combining-site structures will beretained. These residues may be identified by X-ray crystallography ofthe parent antibody or Fab fragment, thereby identifying thethree-dimensional structure of the antigen-binding site. Also, frameworkresidues involved in antigen binding may potentially be identified basedon previously reported humanized murine antibody sequences. Thus, it maybe beneficial to retain such framework residues or others from theparent murine antibody to optimize TAG-72 binding. Preferably, suchmethodology will confer a “human-like” character to the resultanthumanized antibody thus rendering it less immunogenic while retainingthe interior and contacting residues which affect antigen-binding.

The present invention further embraces variants and equivalents whichare substantially homologous to the humanized antibodies and antibodyfragments set forth herein. These may contain, e.g., conservativesubstitution mutations, i.e. the substitution of one or more amino acidsby similar amino acids. For example, conservative substitution refers tothe substitution of an amino acid with another within the same generalclass, e.g., one acidic amino acid with another acidic amino acid, onebasic amino acid with another basic amino acid, or one neutral aminoacid by another neutral amino acid. What is intended by a conservativeamino acid substitution is well known in the art.

The phrase “substantially homologous” is used in regard to thesimilarity of a subject amino acid sequence (of an oligo- orpoly-peptide or protein) to a related, reference amino acid sequence.This phrase is defined as at least about 75% Correspondence, i.e. thestate of identical amino acid residues being situated in parallel,between the subject and reference sequences when those sequences are in“alignment,” i.e. when a minimal number of “null” bases have beeninserted in the subject and/or reference sequences so as to maximize thenumber of existing bases in correspondence between the sequences. “Null”bases are not part of the subject and reference sequences; also, theminimal number of “null” bases inserted in the subject sequence maydiffer from the minimal number inserted in the reference sequence. Inthis definition, a reference sequence is considered “related” to asubject sequence where both amino acid sequences make up proteins orportions of proteins which are either αTAG-72 antibodies or antibodyfragments with αTAG-72 binding affinity. Each of the proteins comprisingthese αTAG-72 antibodies or antibody fragments may independently beantibodies or antibody fragments or hi- or multi-functional proteins,e.g., such as fusion proteins, bi- and multi-specific antibodies, singlechain antibodies, and the like.

The present invention is further directed to nucleic acid sequences fromsuch humanized antibodies may be expressed, as well as expressionvectors which provide for the production of such humanized antibodies intransformed host cells.

In the preferred embodiments, such humanized antibodies andcorresponding nucleic acid sequences will be derived from CC49. Mostpreferably, the humanized heavy chains will have the amino acidsequences set forth in FIG. 1 or 3 and the humanized light chains willhave the amino acid sequences set forth in FIG. 2 or 4. However, asdiscussed, the invention further contemplates other modifications ofthese humanized variable heavy and light sequences, e.g., sequenceswhich further comprise one or more conservative amino acid substitutionsor sequences which retain one or more additional murine frameworkresidues which affect (enhance) antigen binding, which are alternativesto or supplements for those already shown in these Figures.

The subject humanized antibodies, because they specifically bind TAG-72,a pancarcinoma antigen expressed on many different cancer cell types(e.g., colon carcinoma, breast carcinoma, ovarian carcinoma, prostatecarcinoma), and further because they are expected to be significantlynon-immunogenic in humans, should be suitable for use as therapeuticsfor the treatment or prevention of cancers characterized by TAG-72expression, and as diagnostic agents, e.g., for use in tumor imaging orin the RIGS system (Radioimmunoguided Surgery system of Neoprobe Corp.,Dublin, Ohio). See Hinkle et al, Antibody, Immunoconjugates andRadiopharmaceuticals, 4(3):339-358 (1991). One skilled in the art wouldbe able, by routine experimentation, to determine what an effective,non-toxic amount of antibody would be for the purpose of treatingcancer. Generally, however, an effective dosage will be in the range ofabout 0.05 to 100 milligrams per kilogram body weight per day.

The antibodies of the invention may be administered to a mammal inaccordance with the aforementioned methods of treatment in an amountsufficient to produce such effect to a therapeutic, prophylactic, ordiagnostic effect. Such antibodies of the invention can be administeredto such mammal in a conventional dosage form prepared by combining theantibody of the invention with a conventional pharmaceuticallyacceptable carrier or vehicle, diluent, and/or excipient according toknown techniques to form a suspension, injectable solution, or otherformulation. It will be recognized by one of skill in the art that theform and character of the pharmaceutically acceptable carrier or diluentis dictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.

Pharmaceutically acceptable formulations may include, e.g., a suitablesolvent, preservatives such as benzyl alcohol if desired, and a buffer.Useful solvent may include, e.g., water, aqueous alcohols, glycols, andphsophonate and carbonate esters. Such aqueous solutions contain no morethan 50% by volume of organic solvent. Suspension-type formulations mayinclude a liquid suspending medium as a carrier, e.g., aqueouspolyvinylpyrrolidone, inert oils such as vegetable oils or highlyrefined mineral oils, or aqueous cellulose ethers such as aqueouscarboxymethylcellulose. A thickener such as gelatin or an alginate mayalso be present, one or more natural or synthetic surfactants orantifoam agents may be used, and one or more suspending agents such assorbitol or another sugar may be employed therein. Such formations maycontain one or more adjuvants.

The route of administration of the antibody (or fragment thereof) of theinvention may be oral, parenteral, by inhalation or topical. The termparenteral as used herein includes intravenous, intramuscular,subcutaneous, rectal, vaginal or intraperitoneal administration. Thesubcutaneous, intravenous and intramuscular forms of parenteraladministration are generally preferred. The daily parenteral and oraldosage regimens for employing humanized antibodies of the inventionprophylactically or therapeutically will generally be in the range ofabout 0.005 to 100, but preferably about 0.5 to 10, milligrams perkilogram body weight per day.

The antibody of the invention may also be administered by inhalation. By“inhalation” is meant intranasal and oral inhalation administration.Appropriate dosage forms for such administration, such as an aerosolformulation or a metered dose inhaler, may be prepared by conventionaltechniques. The preferred dosage amount of a compound of the inventionto be employed is generally within the range of about 0.1 to 1000milligrams, preferably about 10 to 100 milligrams/kilogram body weight.

The antibody of the invention may also be administered topically. Bytopical administration is meant non-systemic administration. Thisincludes the administration of a humanized antibody (or humanizedantibody fragment) formulation of the invention externally to theepidermis or to the buccal cavity, and instillation of such an antibodyinto the ear, eye, or nose, and wherever it does not significantly enterthe bloodstream. By systemic administration is meant oral, intravenous,intraperitoneal, subcutaneous, and intramuscular administration. Theamount of an antibody required for therapeutic, prophylactic, ordiagnostic effect will, of course, vary with the antibody chosen, thenature and severity of the condition being treated and the animalundergoing treatment, and is ultimately at the discretion of thephysician. A suitable topical dose of an antibody of the invention willgenerally be within the range of about 1 to 100 milligrams per kilogrambody weight daily.

Formulations

While it is possible for an antibody or fragment thereof to beadministered alone, it is preferable to present it as a pharmaceuticalformulation. The active ingredient may comprise, for topicaladministration, from 0.001% to 10% w/w, e.g., from 1% to 2% by weight ofthe formulation, although it may comprise as much as 10% w/w butpreferably not in excess of 5% w/w and more preferably from 0.1% to 1%w/w of the formulation. The topical formulations of the presentinvention, comprise an active ingredient together with one or moreacceptable carrier(s) therefor and optionally any other therapeuticingredients(s). The carrier(s) must be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required, such as liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear, or nose. Drops according to the present invention maycomprise sterile aqueous or oily solutions or suspensions and may beprepared by dissolving the active ingredient in a suitable aqueoussolution of a bactericidal and/or fungicidal agent and/or any othersuitable preservative, and preferably including a surface active agent.The resulting solution may then be clarified and sterilized byfiltration and transferred to the container by an aseptic technique.Examples of bactericidal and fungicidal agents suitable for inclusion inthe drops are phenylmercuric nitrate or acetate (0.002%), benzalkoniumchloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solventsfor the preparation of an oily solution include glycerol, dilutedalcohol and propylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy basis. The basis may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives, or a fattyacid such as stearic or oleic acid together with an alcohol such aspropylene glycol or macrogels. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurface active such as sorbitan esters or polyoxyethylene derivativesthereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

Kits according to the present invention include frozen or lyophilizedhumanized antibodies or humanized antibody fragments to bereconstituted, respectively, by thawing (optionally followed by furtherdilution) or by suspension in a (preferably buffered) liquid vehicle.The kits may also include buffer and/or excipient solutions (in liquidor frozen form)—or buffer and/or excipient powder preparations to bereconstituted with water—for the purpose of mixing with the humanizedantibodies or humanized antibody fragments to produce a formulationsuitable for administration. Thus, preferably the kits containing thehumanized antibodies or humanized antibody fragments are frozen,lyophilized, pre-diluted, or pre-mixed at such a concentration that theaddition of a predetermined amount of heat, of water, or of a solutionprovided in the kit will result in a formulation of sufficientconcentration and pH as to be effective for in vivo or in vitro use inthe treatment or diagnosis of cancer. Preferably, such a kit will alsocomprise instructions for reconstituting and using the humanizedantibody or humanized antibody fragment composition to treat or detectcancer. The kit may also comprise two or more component parts for thereconstituted active composition. For example, a second componentpart—in addition to the humanized antibodies or humanized antibodyfragments—may be bifunctional chelant, bifunctional chelate, or atherapeutic agent such as a radionuclide, which when mixed with thehumanized antibodies or humanized antibody fragments forms a conjugatedsystem therewith. The above-noted buffers, excipients, and othercomponent parts can be sold separately or together with the kit.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of a humanized antibody orhumanized antibody fragment of the invention will be determined by thenature and extent of the condition being treated, the form, route andsite of administration, and the particular animal being treated, andthat such optima can be determined by conventional techniques. It willalso be appreciated by one of skill in the art that the optimal courseof treatment, i.e., the number of doses of an antibody or fragmentthereof of the invention given per day for a defined number of days, canbe ascertained by those skilled in the art using conventional course oftreatment determination tests.

The subject humanized antibodies may also be administered in combinationwith other anti-cancer agents, e.g., other antibodies or drugs. Also,the subject humanized antibodies or fragments may be directly orindirectly attached to effector having therapeutic activity. Suitableeffector moieties include by way of example cytokines (IL-2, TNF,interferons, colony stimulating factors, IL-1, etc.), cytotoxins(Pseudomonas exotoxin, ricin, abrin, etc.), radionuclides, such as ⁹⁰Y,¹³¹I, ^(99m)Tc, ¹¹¹In, ¹²⁵I, among others, drugs (methotrexate,daunorubicin, doxorubicin, etc.), immunomodulators, therapeutic enzymes(e.g., beta-galactosidase), anti-proliferative agents, etc. Theattachment of antibodies to desired effectors is well known. See, e.g.,U.S. Pat. No. 5,435,990 to Cheng et al. Moreover, bifunctional linkersfor facilitating such attachment are well known and widely available.Also, chelators (chelants and chelates) providing for attachment ofradionuclides are well known and available.

Alternatively, the subject humanized antibodies or fragments specific toTAG-72 may be used as immunodiagnostic agents both in vivo and in vitro.A particularly preferred usage is for in vivo imaging of cancer celllesions which express TAG-72. The subject antibodies are preferredbecause they should elicit no significant HAMA or allergic response.Thus, they may be used repeatedly to monitor the disease status of apatient.

As noted above, another preferred application of the subject humanizedantibodies or fragments thereof is in the RIGS system (RadioimmunoguidedSurgery system of Neoprobe Corp., Dublin, Ohio). This technique, alsoknown as the RIGS system involves the intravenous administration of aradiolabeled monoclonal antibody or its fragment prior to surgery. Afterallowing for tumor uptake and blood clearance of radioactivity, thepatient is taken to the operating room where surgical exploration iseffected with the aid of a hand-held gamma activity probe, e.g.,NEOPROBE 1000 (Neoprobe Corp., Dublin, Ohio). This helps the surgeonidentify the tumor metastases and improve the complications of excision.The RIGS system (Radioimmunoguided Surgery system of Neoprobe Corp.,Dublin, Ohio) is advantageous because it allows for the detection oftumors not otherwise detectable by visual inspection and/or palpation.

See, O'Dwyer et al, Arch. Surg., 121:1 391-1394 (1986). This techniqueis described in detail in Hinkle et al, Antibody, Immunoconjugates andRadiopharmacouticals, 4:(3)339-358 (1991) (citing numerous referencesdescribing this technique). This reference also discloses the use ofthis technique with the CC49 monoclonal antibody itself. This techniqueis particularly useful for cancers of the colon, breast, pancreas, andovaries.

The subject humanized antibodies or humanized antibody fragments thereofradiolabeled with radionuclides which are suitable for in vivoadministration, e.g., iodine radionuclides such as ¹³¹I and ¹²⁵I; ¹¹¹Inand ^(99m)Tc are also suitable radiolabels.

The subject humanized antibodies may be used alone or in combinationwith other antibodies. Also, the subject humanized antibodies may beprepared in the form of a diagnostically effective composition.Generally, this will entail the incorporation of diagnosticallyacceptable carriers and excipients, and labels which provide fordetection. Suitable labels include diagnostic radionuclides, enzymes,etc. Methods for using antibodies for tumor imaging are well known inthe art.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The invention will be further clarified by aconsideration of the following examples, which are intended to be purelyexemplary of the present invention are thus to be construed as merelyillustrative examples and not limitations of the scope of the presentinvention in any way.

EXAMPLES Materials and Methods

DNA Template Preparation

All recombination work was performed upon DNA sequences in plasmid M13vectors. The source of the NEWM framework regions for producing theinitial humanized CC49 V_(H) was an M13 construct bearing, between theM13 BamHI and HindIII sites a DNA segment having the nucleotide sequenceshown in FIG. 13. The source of REI framework regions for producing theinitial humanized CC49 VL was an M13 construct bearing—between the M13BamHI and HindIII sites—a DNA segment encoding the REI amino acidsequence of FIG. 2.

When overlap-extension procedures were used to introduce mutations intoa given DNA sequence, double stranded M13 DNA was utilized. In contrast,when extension-ligation procedures were used instead, theoligonucleotides were designed to anneal to only one of the two DNAstrands. In this latter procedure, the M13 DNA was first treated tosubstitute uridine for every thymidine base in the DNA, to produceuridinylated DNA. This was accomplished by transfecting the M13 plasmidDNA into competent cells lacking dUTPase and uracil glycosylase,normally RZ1032 cells (though CJ236 cells available from Bio-Rad ofHercules, Calif., are also suitable) by combining the followingingredients.

1 μL of M13 plasmid DNA

4 mL of LB broth

40 μL of competent RZ1032 cells.

The culture was shaken for 5 hours at 37° C. and the resultingsingle-stranded plasmid DNA (ssDNA) was isolated and dissolved in 50 μLTris-EDTA buffer. The DNA was then treated with uracil glycosylase bymixing together:

1 μL uridinylated ssDNA

1 μL 10×glycosylase buffer

1 U uracil glycosylase (Gibco BRL, Gathersburg, Md.)

40 μL 25 mM MgCI2.

This mixture was then incubated at 37° C. for one hour and then 6.6 μL25 mM MgCI2 and 9.9 μL 1M NaOH. The mixture was then further incubatedfor 5 minutes at 37° C. and 16.5 μL of 0.6M HCI was then added toneutralize the mixture. The DNA was then ethanol precipitated anddissolved in water.

M13 Oligonucleotide Primers

The following oligonucleotide primers were used throughout the processof preparing the humanized CC49 VHs and VLs exemplified below.

10. 5′-CTAAAACGACGGCCAGT-3′ (SEQ ID NO:18);

11. 5′-AACAGCTATGACCATG-3′ (SEQ ID NO:19);

385. 5′-GCGGGCCTCTTCGCTATTACGC-3′ (SEQ ID NO:20); and

391. 5′-CTCTCTCAGGGCCAGGCGGTGA-3′ (SEQ ID NO:21).

These primers are complementary to regions of the plasmid M13 which areexternal both to the (NEWM or REI) target framework sequences and to theBamHI site-to-HindIII site section of M13.

Murine Variable-Regions

In order to compare the antibody binding characteristics of theantibodies produced according to the examples set forth below,antibodies having a chimeric heavy chain (i.e. a heavy chain having amurine CC49 VH region and a human IgG1 constant region) and/or achimeric light chain (i.e. a light chain having a murine CC49 VL and ahuman κ constant region) were expressed. The source of these chimericchains was the ATCC-deposited cell line HB9884 (Budapest) whichexpresses a chimeric CC49 antibody having both chains. The heavy chainof this antibody was termed “MuVH” and the light chain thereof wastermed “MuVL.”

Oligonucleotide Phosphorylation Protocol

Mutating oligonucleotides used in non-overlap extensions werephosphorylated according to the procedure below. In a final volume of 25μL, the following ingredients were combined:

10 pmol of each oligonucleotide,

5 μL of a 5×polynucleotide kinase buffer, and

5 U of T4 polynucleotide kinase (Gibco BRL).

The phosphorylation reaction was started with the addition of the enzymeand allowed to proceed for one hour at 37° C.

Annealing Protocol for Non-Overlap Extension-ligations

The annealing step for non-overlap extension-ligations involvedperforming one annealing in which all mutation-carrying oligonucleotidesand one primer oligonucleotide were annealed to a single stranded DNAtemplate in which all thymidine bases had been replaced with uridinebases. The mutating oligonucleotides were first phosphorylated accordingto the above oligonucleotide phosphorylation protocol. In a final volumeof 20 μL, the following ingredients were combined:

1 pmol of each mutation-carrying phosphorylated oligonucleotide

1 pmol of a primer oligonucleotide

4 μL 5×annealing buffer

0.2 pmol ssU-DNA template.

The mixture was then heated to 90° C. for 30 sec., then quickly cooledto 70° C., and finally allowed to slowly cool to 37° C.

Extension-ligation Protocol for Non-overlap Extension-ligations

After completion of the annealing step in which the primer andphosphorylated mutating oligonucleotides were annealed to the ssU-DNAtemplate, extension-ligation was performed as follows. In a final volumeof 30 μL, the following ingredients were combined:

20 μL annealed ssU-DNA (i.e. the contents of the above annealingprocedure)

2 μL 5×annealing buffer

2 μL 0.1 M dithiothreitol

0.3 μL 0.1M ATP

1 μL 6.25 mM dNTP mixture of equimolar amounts of dATP, dTTP, dGTP, dCTP

2.5 U T7 DNA polymerase (USB, now Amersham Life Sciences, Cleveland,Ohio)

0.5 U T4 DNA ligase (Gibco BRL)

Water to 30 μL.

This mixture was then incubated at room temperature for 1 hour.

Standard PCR Protocols

The following procedure was used, alternately, both to amplify thenon-overlap extension-ligation DNA sequences and to perform extension ofeach overlap DNA sequence. In a final volume of 50 μL, the followingingredients were combined:

2 μL template DNA (either annealed ssU-DNA or non-annealed ssDNA)

5 μL 10×Vent buffer (NEB, i.e. New England Biolabs, Beverly, Mass.) or10×Thermalase buffer (IBI of New Haven, Conn.)

2 μL 6.25 mM dNTP mixture of equimolar amounts of dATP, dTTP, dGTP, dCTP

25 pmol of one oligonucleotide primer

25 pmol of either a mutation-carrying oligonucleotide (foroverlap-extension) or a second oligonucleotide primer

1 U Vent DNA polymerase (NEB) or Thermalase DNA polymerase (IBI).

Reactions were initiated with the addition of the DNA polymerase andthen treated with about 15 cycles of: (1) 94° C. for 30 sec., (2) 50° C.for 30 sec., and (3) 30-60 seconds at either 75° C. (for Vent DNApolymerase) or 72° C. (for Thermalase). Reactions were brought tocompletion with 5 minutes at a constant temperature of either 75° C. ForVent DNA polymerase) or 72° C. (for Thermalase).

PCR Overlap-extension Amplification Protocol

After a pair of PCR reactions were performed—one for each of the two(partially complementary) overlapping DNA segments—the two resultingsegments were joined according to the following PCR procedure. In afinal volume of 50 μL, the following ingredients were mixed:

1 μL of each overlap DNA (from the above overlap PCR extensionreactions)

5 μL 10×Vent buffer (NEB) or Thermalase buffer (IBI)

2 μL 6.25 mM dNTP mixture of equimolar amounts of dATP, dTTP, dGTP, dCTP

25 pmol of each oligonucleotide primer used in the overlap PCRextensions

1 U Vent DNA polymerase (NEB) or Thermalase DNA polymerase (IBI).

Reactions were initiated with the addition of the DNA polymerase andthen treated with about 15 cycles of: (1) 94° C. for 30 sec., (2) 50° C.for 30 sec., and (3) 30-60 seconds at either 75° C. (for Vent DNApolymerase) or 72° C. (for Thermalase). Reactions were brought tocompletion with 5 minutes at a constant temperature of either 75° C.(for Vent DNA polymerase) or 72° C. (for Thermalase).

Transfer of Humanized CC49 Variable Region DNA Sequences from M13 to pSVVectors and Subsequent Antibody Expression

Humanized antibodies were expressed in pSV vectors grown in NSO cells.The humanized variable region constructs which were produced in theplasmid, M13, were digested with 10 U each of HindIII and BamHI (bothfrom BRL, i.e. Gibco BRL) for 1 hour at 37° C. in a final volume of 100μL with Tris-EDTA buffer. The resulting DNA fragments were then run on alow melting point agarose gel, the band containing the humanizedconstruct DNA was cut out, and the DNA was purified using an ELUTIP ‘d’column with 20 μL Tris-EDTA buffer. 10 μL of the purified DNApreparation was then combined with 1 μL of a HindIII and BamHI-digestedpSV preparation, 3 μL of 5× ligase buffer, and 1 U of T4 DNA ligase(BRL), in order to insert the construct into a pSV plasmid. HumanizedCC49 VH constructs were inserted into pSVgpt vectors bearing a humanIgG1 heavy chain constant region; the pSVgpt vector used is the“aLYS-30” shown in FIG. 5. Humanized CC49 VL constructs were insertedinto pSVhyg vectors bearing a human κ light chain constant domain; thepSVhyg vector used in the “aLys-17” shown in FIG. 5. Each humanizedvariable region construct was inserted adjacent to the respectiveconstant region, i.e. so as to replace either the HuVHLYS or the HuVLLyssegment illustrated in FIG. 5.

The resulting vectors were transfected into NSO cells as follows. About3 μg of the VH vector, or about 6 μg of the VL vector, produced by thepSV-insertion procedures, was then linearized by digestion with 10 UPvul (Gibco BRL). The digested DNA was then precipitated with ethanoland redissolved in 50 μL of water. NSO cells were collected bycentrifugation and resuspended in 0.5 mL Dulbecco's Modified Eagle'sMedium (DMEM) and then transferred to a Gene Pulser cuvette (Bio-Rad).The DNA from both one VH and one VL construct was gently mixed with thecells by pipetting and the cuvette was left on ice for 5 minutes. Next,the cuvette was inserted between the electrodes of the Bio-Rad GenePulser and a single pulse of 170V at 960 μF was applied. The contents ofthe cuvette were then transferred to a flask containing 20 mL DMEM andthe cells were allowed to rest for 1-2 days at 37° C. Cells were againharvested by centrifugation and resuspended in 36 mL selective DMEM. 1.5mL aliquots of this resuspension were placed in each well of a 24-wellplate and incubated at 37° C. for 4 days, at which time the medium ineach well was replaced with 1.5 mL of fresh selective DMEM. After 6 moredays of incubation at 37° C., surviving cell colonies were visible tothe naked eye and the supernatants of each well were assayed forantibody production. Both whole antibody production (i.e. withoutpurification) and purified antibody production were assayed. To obtainpurified antibodies, the supernatants were passed through a protein Acolumn.

ELISA Assay Protocols

Antibody concentrations and antibody binding characteristics were testedusing enzyme-linked immunosorbent assay (ELISA) procedures which are setforth as follows.

Measurement of IgG Concentration

The concentration of IgG secreted from transfected cells was measuredusing an enzyme-linked immunosorbent assay (ELISA) procedure which isset forth as follows.

Polyvinyl chloride (PVC) microtiter plates (Dynatech Laboratories,Chantilly, Va., catalog # 001-010-2101) were coated with goat anti-humanIgG (0 mg/mL, GAHIG, Southern Biotechnology Associates, Inc.,Birmingham, Ala., catalog # 2010-01) diluted with Milli-Q® water andplaced on the plates using 50 mL/well. Plates were air-dried overnightat ambient temperature or at 37° C. for 3 hours. Prior to use,non-specific binding was blocked the addition of 0.2 mL/well of 1% (w/v)bovine serum albumin (Sigma, St. Louis, Mo. catalog # A7888) inphosphate buffered saline (Sigma, catalog # 1000-3) (PBS/BSA). Allincubations were carried out in a humidified container. Plates wereincubated for 1-2 hours at 37° C. and the blocking solution removedprior to sample addition. Two-fold serial dilutions of samples or astandard IgG solution set at 500 ng/mL (50 μL/well) were made intriplicate in the PBS/BSA solution. The plate was incubated at 37° C.for 3 hours or overnight at 4° C. The plate was washed 3 times with0.025% Tween-20 (v/v, Sigma) using an automatic plate washer. 50 μL/wellof 1:1000 dilution of a goat anti-human IgG conjugated to HorseradishPeroxidase (Southern Biotechnology Associates Inc.) was added andincubated at 37° C. for 1.5 hours. The wells were washed 3 times with0.025% Tween-20 (v/v, Sigma) using an automatic plate washer and 50μL/well OPD substrate buffer added. The color was developed for 4minutes, stopped with 12.5 μL 12.5% H₂SO₄ and the absorbance at 492 nmread. The concentration of IgG in the test sample was estimated bycomparison of the mean of the optical densities to a standard curveconstructed from the standard IgG.

Determination of Relative Affinities of Humanized Antibodies

Antibody binding characteristics were tested in an ELISA using partiallypurified TAG-72 antigen immobilized on Polyvinyl chloride (PVC)microtiter plates (Dynatech Laboratories, Chantilly, Va., catalog#001-010-2101)

PVC plates were coated with 50 μL/well TAG-72 (Dow Chemical, lot#040191), diluted 1:300 in Milli-Q water. Plates were air-driedovernight at ambient temperature or at 37° C. for 3 hours. Prior to use,non-specific binding was blocked the addition of 0.2 mL/well of 1% (w/v)bovine serum albumin (Sigma, St. Louis, Mo. catalog # A7888) inphosphate buffered saline (Sigma, catalog # 1000-3) (PBS/BSA). Plateswere incubated for 1-2 hours at 37° C. and the blocking solution removedprior to sample addition. All incubations were carried out in ahumidified container. Two-fold serial dilutions (starting concentrationrange of 1.0 g/ml-10 μg/ml) of the samples to be tested in the PBS/BSAsolution were added to triplicate wells of the TAG-coated plate (50μL/well). The plate was incubated overnight at 4° C. or 1-2 hours at 37°C. The plate was washed 3-times with 0.025% Tween-20 (v/v, Sigma) usingan automatic plate washer. 50 μL/well of 1:1000 dilution of a goatanti-human IgG conjugated to Horseradish Peroxidase (SouthernBiotechnology Associates Inc.) was added and incubated at 37° C. for 1.5hours. The wells were washed 3 times with 0.025% Tween-20 (v/v, Sigma)using an automatic plate washer and 50 μL/well OPD substrate bufferadded. The color was developed for 4 minutes, stopped with 12.5 μL 12.5%H₂SO₄ and the absorbance at 492 nm read.

Determination of Affinity Constants for Binding to TAG-72

Two-fold dilutions of purified Hu-CC49 were prepared in PBS/BSA over arange of 1.0 μg/ml-0.003 μg/ml and samples (20 uL/well) were applied intriplicate to TAG coated PVC prepared and blocked as described supra.Plates were incubated overnight at 4° C. Following this incubation,samples were transferred from the plate to the corresponding wells onthe GAHIG-coated trap plate. The original TAG plate was washed 3-timeswith 0.025% Tween-20 (v/v, Sigma, catalog # P1379) using an automaticplate washer. An ¹²⁵I-labeled goat anti-human IgG probe (ICNBiomedicals, Inc., catalog #68088) was diluted to 75,000 cpm/25μL inPBS/BSA and added (25 μL/well) to all wells. This TAG plate wasincubated for 1 hour at 37° C.

After a 1 hour incubation at 37° C., the trap plate was washed asdescribed above and ¹²⁵I-labeled GAHIG probe was added. This plate wasincubated for 1 hour at 37° C. Both plates (TAG and GAHIG-trap)containing probe were then washed with a microplate washer to remove theunbound probe. A plate cutter (D. Lee, Sunnyvale, Calif., Model HWC-4)was used to separate the wells from the plate frame. The radioactivityin each well was quantified by a gamma counter. The resulting data wasanalyzed according to the method of Scatchard (Ann. NY Acad., 51:600-672(1946)).

Example 1 Preparation of CDR-grafted (Initial Humanized) Antibody FromMurine CC49

We describe in this Example the construction of humanized CC49 Mabs(CC49 HuVH/HuVK) using the V_(L) and V_(H) frameworks of human Mabs REIand NEWM, respectively. The CDRs for murine CC49 were grafted onto humanframeworks according to known methods as discussed supra. In particular,human frameworks were selected from antibodies which, based on previousstudies, were predicted to be suitable, i.e. which should not adverselyaffect antigen binding and not exhibit significant immunogenicity inhumans. The human frameworks selected for the variable heavy andvariable light chains, respectively, were NEWM and REI. In theproduction of the initial version of the humanized VH, certain murineframework residues were also retained which, based on previous studies,might allow retention of antigen binding properties. Specifically,residues Y27, T30, A72, F95, and T97 of the murine heavy chain wereinitially retained. Concurrently, an alternate version of the humanizedVH was produced which retained, in addition, the murine frameworkresidue A24.

The production of these NEWM-grafted humanized CC49 VHs was accomplishedaccording to the annealing and extension-ligation protocols describedabove, using a single-stranded M13 DNA template bearing, between theHindIII and BamHI sites thereof, a DNA segment having the nucleotidesequence shown in FIG. 13. In this procedure, Primer 11 was used inconjunction with a set of mutating oligonucleotides. These mutatingoligonucleotides were designed and synthesized with the followingsequences:

1a. 5′-GCTGTCTCACCCAGTGAATTGCATGGTCAGTGAAGGTGTAGCCAGA CACGGTGCAGGTCA-3′(SEQ ID NO:22);

1b. 5′-GCTGTCTCACCCAGTGAATTGCATGGTCAGTGAAGGTGTAGCCAGA CGCGGTGCAGGTCA-3′(SEQ ID NO:23);

2. 5′-CTGGTGTCTGCCAGCATTGTCACTCTCCCCTTGAACCTCTCATTGTATTTAAAATCATCATTTCCGGGAGAAAAATATCCAATCCACTCAAGAC-3′ (SEQ ID NO:24); and

3. 5′-GGACCCTTGGCCCCAGTAGGCCATATTCAGGGATCTTGTACAGAAAT AGACCGCGGTGTC-3′(SEQ ID NO:25)

Codons which were designed into the oligonucleotides in order to retainmurine FR amino acids are shown in bold-face type. Afterextension-ligation, amplifying PCR was performed using the standard PCRprotocol with Vent DNA polymerase. The use of two versions of mutatingoligonucleotide 1 resulted in the formation of two initial humanizedV_(H) ^(_(S)) . These were named “CC49 NMVH,” also called “HuVH,” (forconstructs incorporating oligonucleotide 1) and “HuVHA” (for constructsincorporating oligonucleotide 1b).

In the production of the initial version of the humanized VL, nouniquely murine framework residues were retained. The production of theREI-grafted humanized CC49 V, was accomplished according to theannealing and extension-ligation protocols described above, using assM13 template bearing, between the HindIII and BamHI sites thereof, assU-DNA segment encoding the REIVK sequence shown in FIG. 2. In thisprocedure, Primer 385 was used in conjunction with a set of mutatingoligonucleotides. These mutating oligonucleotides were designed andsynthesized with the following sequences:

21. 5′-GTTCTTCTGATTACCACTGTATAAAAGACTTTGACTGGAC-3′ (SEQ ID NO:26);

22. 5′-CAGATTCCCTAGCGGATGCCCAGTAG-3′ (SEQ ID NO:27);

23. 5′-TTCTACTCACGTGTGATTTGCAGCTTGGTCCCTTGGCCGAACGTGAGGGGATAGGAATAGTATTGCTGGCAGTAGTAG-3′ (SEQ ID NO:28); and

24. 5′-GCTCTGGGTCATCTGGATGTCGG-3′ (SEQ ID NO:29).

After extension-ligation, amplifying PCR was performed using theThermalase standard PCR protocol described above. The resultinghumanized V_(L) was named “CC49 REVK” and was also called “HuVK.”

The two heavy chain constructs, HuVH and HuVHA, and the light chainconstruct, HuVH, which were situated in M13 vectors, were grown andexpressed in TG1 cells. The polypeptide expression products of theseconstructs were sequenced and the amino acid sequences of theseconstructs are presented in FIGS. 1 and 2.

These DNA constructs were then inserted into pSV expression vectors asdescribed above. Combinations of these with each other or with the ATCC(Budapest) HB 9884 DNA sequences encoding the chimeric MuVH or MuVL werethen inserted into NSO cells. Specifically, the following fourcombinations of heavy and light chain constructs were separatelytransfected into NSO cells as described above: HuVHA and MuVK, HuVHA andHuVK, MuVH and MuVK, and HuVH and HuVK. These combinations wereexpressed and the resulting antibodies were then tested for antigenbinding characteristics using the ELISA assay set forth above. Theresults of these assays are shown in FIGS. 6-9.

The data in FIG. 6 show that, with HuVHA, the HuVK humanized light chainfunctions as well as the MuVK chimeric light chain. FIGS. 7 and 8indicate that the fully humanized HuVH/HuVK antibody binds TAG-72approximately 2-fold less than the chimeric MuVH/MuVK antibody. Also,FIG. 9 suggests that the A24 mutation produces no enhancement in antigenbinding; rather, the A24 mutation causes an approximate 2-fold reductionin affinity as measured by this ELISA assay.

Further Development of Humanized CC49

Because it appears that the HuVK humanized light chain functions as wellas the MuVK chimeric light chain, further work was directed to makingalternate mutated versions of the HuVH humanized heavy chain. A firstvariant of HuVH was made by replacing the Lysine residue shown atposition 76 of the CC49 NMVH in FIG. 1 with the murine FR residue,serine. This was achieved by the Vent DNA polymerase PCR overlapextension protocol described above, using two primer-and-mutatingoligonucleotide pairs—oligonucleotides 10 and 4, and oligonucleotides 11and 5. Each pair was used in conjunction with one of the strands of adsDNA template—situated between the HindIII and BamHI sites of plasmidM13—and having the nucleotide sequence shown in FIG. 14 (the upperstrand was used with pair 11 & 5). Mutating oligonucleotides 4 and 5were designed and synthesized with the following sequences:

4. 5′-AGACACCAGCAGCAACCAGTTCAG-3′(SEQ ID NO:30); and

5. 5′-GCTGAACTGGTTGCTGCTGGTGTC-3′(SEQ ID NO:3 1).

The serine residue codons are shown in bold-face type. The resultinghumanized CC49 V_(H) was named “HuVHS.”

A second variant of HUVH was made by replacing the threonine residueshown at position 74 of the CC49 NMVH in FIG. 1 with the murine FRresidue, Lysine.

This was achieved by the Vent DNA polymerase PCR overlap extensionprotocol described above, using two primer-and-mutating oligonucleotidepairs—oligonucleotides 10 and 6, and oligonucleotides 11 and 7. Eachpair was used in conjunction with one of the strands of a dsDNAtemplate—situated between the HindIII and BamHI sites of plasmid M13—andhaving the nucleotide sequence shown in FIG. 14 (the upper strand wasused with pair 11 & 7). Mutating oligonucleotides 6 and 7 were designedand synthesized with the following sequences.

6. 5′-CTGGCAGACAAGAGCAAGAACCAG-3′ (SEQ ID NO:32).

7. 5′-TGGTTCTTGCTCTTGTCTGCCAGC-3′(SEQ ID NO:33).

The Lysine residue codons are shown in bold-face type. The resultinghumanized CC49 VH was named “HuVHK.”

The two heavy chain constructs, HUVHS and HUVHK, which were situated inM13 vectors, were grown and expressed in TG1 cells. The polypeptideexpression products of these constructs were sequenced and the aminoacid sequences of these constructs are presented in FIG. 1.

These DNA constructs were then inserted into pSV expression vectors asdescribed above. Combinations of these with HUVK were then inserted intoNSO cells. These combinations were expressed and the resultingantibodies were then tested for antigen binding characteristics usingthe ELISA assay set forth above. The results of these assays are shownin FIGS. 10 and 11. These figures show that neither the K74 nor S76mutation resulted in enhanced antigen binding; in fact the S76 mutationcaused an approximate 2-fold reduction in affinity as measured by thisELISA assay.

Example 2 Measurement of Affinity Constant for the Humanized CC49Antibody Obtained in Example 1

The affinity constant of the final humanized CC49 antibody, CC49HuVH/HuVK, of Example 1 (having variable heavy and variable light chainsequences shown in FIGS. 3 and 4, respectively) were measured accordingto the ELISA assay protocol set forth above. The ATCC (Budapest) HB 9884chimeric CC49 antibody was used as an internal control. This ELISA assaywas performed repeatedly using purified samples of humanized andchimeric CC49 monoclonal antibodies in order to substantiate theaccuracy of the values obtained and to account for inherentassay-to-assay variability. Typical results from such an analysis areshown in FIG. 12. A summary of the results obtained during theseanalyses is shown in Table 1.

TABLE 1 Summary of Affinity Constant Analysis of Humanized & ChimericCC49 Chimeric CC49¹ CC49 HuVH/HuVK Affinity Constant (Ka) 7.62 × 10⁹M⁻¹4.27 × 10⁹M⁻¹ ± standard deviation 3.94 × 10⁹M⁻¹ 2.57 × 10⁹M⁻¹ number ofanalyses 7 8

These results demonstrate that the humanized anti-TAG-72 antibodyHuVH/HuVK (having the variable heavy and variable light sequences shownin FIGS. 3 and 4, respectively) has a binding affinity approximatingthat of a chimeric CC49 antibody (MuVH/MuVK). Therefore, it is expectedthat this humanized antibody will effectively target TAG-72-expressingcarcinomas in vivo. Also, based on its sequence this antibody shouldexhibit little or no immunogenicity in humans, and exhibit advantageousplasma clearance, metabolic properties, and effective tumor targeting inrelation to the murine CC49, and also in relation to chimeric versionsthereof.

A murine plasmacytoma cell line which produces this humanized CC49antibody was deposited with the American Type Culture Collection (10801University Boulevard, Manassas, Va. 20110-2209, on Oct. 16, 1996) andthis cell line was accorded accession number ATCC CRL-12209. Thisdeposit was made in accordance with the Budapest Treaty. This depositedcell line will be made irrevocably available, without restriction, uponissuance of a patent to this application or any other applicationclaiming priority to this application under 35 U.S.C. § 120.

Based on the foregoing, it will be appreciated that the humanizedantibodies disclosed in Examples 1-3, exhibit antigen-bindingcharacteristics, i.e. TAG-72 affinities comparable to the parentmonoclonal antibody, nCC49 (murine antibody), and to chimeric antibodiesderived from nCC49, e.g., cCC49. Moreover, based on the foregoingresults, these antibodies possess properties which will render them wellsuited for usage as in vivo diagnostics or therapeutics, e.g., improvedserum clearance, metabolic properties, and little or no immunogenicityin humans.

These properties are highly significant because these properties willenable the subject humanized antibodies to be administered repeatedly,in large dosages, and over a prolonged period of time withoutsignificant adverse effects, e.g., a HAMA response or non-specificcytotoxicity. This is important for cancer treatment as well as forcancer diagnosis as it enables these antibodies to be used overprolonged time periods. Moreover, the clearance properties of thesubject human antibodies will enable these antibodies to effectivelytarget desired target sites, e.g., TAG-72 expressing carcinomas (becauseof the effects of serum clearance on targeting efficiency). Therefore,the humanized antibodies of the present invention comprise a substantialimprovement in relation to previously disclosed antibodies specific toTAG-72.

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of this specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 33 <210> SEQ ID NO 1 <211>LENGTH: 115 <212> TYPE: PRT <213> ORGANISM: Mus musculus <220> FEATURE:<221> NAME/KEY: Murine CC49 VH <222> LOCATION: 1..115 <400> SEQUENCE: 1Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His 20 25 30Ala Ile His Trp Val Lys Gln Asn Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Phe Ser Pro Gly Asn Asp Asp Phe Lys Tyr Asn Glu Arg Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 7580 Val Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 9095 Thr Arg Ser Leu Asn Met Ala Tyr Trp Gly Gln Gly Thr Ser Val Thr 100105 110 Val Ser Ser 115 <210> SEQ ID NO 2 <211> LENGTH: 115 <212> TYPE:PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: NEWM VHFR template <222> LOCATION: 1..115 <223> OTHER INFORMATION: CDR aminoacids are indicated by Xaa <400> SEQUENCE: 2 Gln Val Gln Leu Gln Glu SerGly Pro Gly Leu Val Arg Pro Ser Gln 5 10 15 Thr Leu Ser Leu Thr Cys ThrVal Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Trp Val Arg GlnPro Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser ValThr Ala Ala Asp Thr Ala Val Tyr Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Trp Gly Gln Gly Ser Leu Val Thr 100 105 110 Val Ser Ser 115<210> SEQ ID NO 3 <211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <221> NAME/KEY: Humanized CC49 VH,HuVH <222> LOCATION: 1..115 <223> OTHER INFORMATION: Heavy chainvariable region containing human NEWM FRs and murine CC49 VH CDRs <400>SEQUENCE: 3 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro SerGln 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr AspHis 20 25 30 Ala Ile His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu TrpIle 35 40 45 Gly Tyr Phe Ser Pro Gly Asn Asp Asp Phe Lys Tyr Asn Glu ArgPhe 50 55 60 Lys Gly Arg Val Thr Met Leu Ala Asp Thr Ser Lys Asn Gln PheSer 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val TyrPhe Cys 85 90 95 Thr Arg Ser Leu Asn Met Ala Tyr Trp Gly Gln Gly Ser LeuVal Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO 4 <211> LENGTH: 115<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221>NAME/KEY: Humanized CC49 VH, HuVHA <222> LOCATION: 1..115 <223> OTHERINFORMATION: Heavy chain variable region containing human NEWM FRs andmurine CC49 VH CDRs, and retaining a murine alanine in FR1 <400>SEQUENCE: 4 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro SerGln 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Tyr Thr Phe Thr AspHis 20 25 30 Ala Ile His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu TrpIle 35 40 45 Gly Tyr Phe Ser Pro Gly Asn Asp Asp Phe Lys Tyr Asn Glu ArgPhe 50 55 60 Lys Gly Arg Val Thr Met Leu Ala Asp Thr Ser Lys Asn Gln PheSer 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val TyrPhe Cys 85 90 95 Thr Arg Ser Leu Asn Met Ala Tyr Trp Gly Gln Gly Ser LeuVal Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO 5 <211> LENGTH: 115<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221>NAME/KEY: Humanized CC49 VH, HuVHS <222> LOCATION: 1..115 <223> OTHERINFORMATION: Heavy chain variable region containing human NEWM FRs andmurine CC49 VH CDRs, and retaining a murine serine in FR3 <400>SEQUENCE: 5 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro SerGln 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr AspHis 20 25 30 Ala Ile His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu TrpIle 35 40 45 Gly Tyr Phe Ser Pro Gly Asn Asp Asp Phe Lys Tyr Asn Glu ArgPhe 50 55 60 Lys Gly Arg Val Thr Met Leu Ala Asp Thr Ser Ser Asn Gln PheSer 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val TyrPhe Cys 85 90 95 Thr Arg Ser Leu Asn Met Ala Tyr Trp Gly Gln Gly Ser LeuVal Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO 6 <211> LENGTH: 115<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221>NAME/KEY: Humanized CC49 VH, HuVHK <222> LOCATION: 1..115 <223> OTHERINFORMATION: Heavy chain variable region containing human NEWM FRs andmurine CC49 VH CDRs, and retaining a murine lysine in FR3 <400>SEQUENCE: 6 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro SerGln 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr AspHis 20 25 30 Ala Ile His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu TrpIle 35 40 45 Gly Tyr Phe Ser Pro Gly Asn Asp Asp Phe Lys Tyr Asn Glu ArgPhe 50 55 60 Lys Gly Arg Val Thr Met Leu Ala Asp Lys Ser Lys Asn Gln PheSer 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val TyrPhe Cys 85 90 95 Thr Arg Ser Leu Asn Met Ala Tyr Trp Gly Gln Gly Ser LeuVal Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO 7 <211> LENGTH: 113<212> TYPE: PRT <213> ORGANISM: Mus musculus <220> FEATURE: <221>NAME/KEY: Murine CC49 VK <222> LOCATION: 1..113 <400> SEQUENCE: 7 AspIle Val Met Ser Gln Ser Pro Ser Ser Leu Pro Val Ser Val Gly 5 10 15 GluLys Val Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 GlyAsn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 SerPro Lys Leu Leu Ile Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val 50 55 60 ProAsp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser 65 70 75 80Ile Ser Ser Val Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Val Leu 100 105110 Lys <210> SEQ ID NO 8 <211> LENGTH: 113 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: REI VK FR template<222> LOCATION: 1..113 <223> OTHER INFORMATION: CDR amino acids areindicated by Xaa <400> SEQUENCE: 8 Asp Ile Gln Leu Thr Gln Ser Pro SerSer Leu Ser Ala Ser Val Gly 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys SerSer Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Lys Asn Tyr Leu Ala TrpTyr Gln Gln Thr Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp AlaXaa Xaa Xaa Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly SerGly Thr Asp Tyr Thr Phe THr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu AspIle Ala Thr Tyr Tyr Cys Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa PheGly Gln Gly Thr Lys Leu Gln Ile 100 105 110 Thr <210> SEQ ID NO 9 <211>LENGTH: 113 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <221> NAME/KEY: Humanized CC49 VK, HuVK <222> LOCATION: 1..113<223> OTHER INFORMATION: Light chain variable region containing humanREI FRs and murine CC49 VL CDRs <400> SEQUENCE: 9 Asp Ile Gln Met ThrGln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 5 10 15 Asp Arg Val Thr IleThr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Gly Asn Gln Lys AsnTyr Leu Ala Trp Tyr Gln Gln Thr Pro Gly Lys 35 40 45 Ala Pro Lys Leu LeuIle Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe SerGly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr 65 70 75 80 Ile Ser Ser LeuGln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser TyrPro Leu Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile 100 105 110 Thr <210>SEQ ID NO 10 <211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM: Musmusculus <220> FEATURE: <221> NAME/KEY: Murine CC49 VH <222> LOCATION:1..115 <400> SEQUENCE: 10 Gln Val Gln Leu Gln Gln Ser Asp Ala Glu LeuVal Lys Pro Gly Ala 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly TyrThr Phe Thr Asp His 20 25 30 Ala Ile His Trp Val Lys Gln Asn Pro Glu GlnGly Leu Glu Trp Ile 35 40 45 Gly Tyr Phe Ser Pro Gly Asn Asp Asp Phe LysTyr Asn Glu Arg Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys SerSer Ser Thr Ala Tyr 65 70 75 80 Val Gln Leu Asn Ser Leu Thr Ser Glu AspSer Ala Val Tyr Phe Cys 85 90 95 Thr Arg Ser Leu Asn Met Ala Tyr Trp GlyGln Gly Thr Ser Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO 11<211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <221> NAME/KEY: HuCC49 VH <222> LOCATION: 1..115 <223>OTHER INFORMATION: Heavy chain variable region containing human NEWM FRsand murine CC49 VH CDRs <400> SEQUENCE: 11 Gln Val Gln Leu Gln Glu SerGly Pro Gly Leu Val Arg Pro Ser Gln 5 10 15 Thr Leu Ser Leu Thr Cys ThrVal Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Ala Ile His Trp Val Arg GlnPro Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Phe Ser Pro Gly AsnAsp Asp Phe Lys Tyr Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Met LeuAla Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser ValThr Ala Ala Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Arg Ser Leu Asn MetAla Tyr Trp Gly Gln Gly Ser Leu Val Thr 100 105 110 Val Ser Ser 115<210> SEQ ID NO 12 <211> LENGTH: 117 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: NEWM VH <222> LOCATION:1..117 <400> SEQUENCE: 12 Gln Val Gln Leu Glu Gln Ser Gly Pro Gly LeuVal Arg Pro Ser Gln 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly SerThr Phe Ser Asn Asp 20 25 30 Tyr Tyr Thr Trp Val Arg Gln Pro Pro Gly ArgGly Leu Glu Trp Ile 35 40 45 Gly Tyr Val Phe Tyr His Gly Thr Ser Asp AspThr Thr Pro Leu Arg 50 55 60 Ser Arg Val Thr Met Leu Val Asp Thr Ser LysAsn Gln Phe Ser Leu 65 70 75 80 Arg Leu Ser Ser Val Thr Ala Ala Asp ThrAla Val Tyr Tyr Cys Ala 85 90 95 Arg Asn Leu Ile Ala Gly Cys Ile Asp ValTrp Gly Gln Gly Ser Leu 100 105 110 Val Thr Val Ser Ser 115 <210> SEQ IDNO 13 <211> LENGTH: 113 <212> TYPE: PRT <213> ORGANISM: Mus musculus<220> FEATURE: <221> NAME/KEY: Murine CC49 VL <222> LOCATION: 1..113<400> SEQUENCE: 13 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Pro ValSer Val Gly 5 10 15 Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gln Ser LeuLeu Tyr Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln LysPro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ala Arg GluSer Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp PheThr Leu Ser 65 70 75 80 Ile Ser Ser Val Lys Thr Glu Asp Leu Ala Val TyrTyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly ThrLys Leu Val Leu 100 105 110 Lys <210> SEQ ID NO 14 <211> LENGTH: 113<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221>NAME/KEY: HuCC49 VL <222> LOCATION: 1..113 <223> OTHER INFORMATION:Light chain variable region containing human REI FRs and murine CC49 VLCDRs <400> SEQUENCE: 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln SerLeu Leu Tyr Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln GlnThr Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ala ArgGlu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr AspTyr Thr Phe Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala ThrTyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Gln GlyThr Lys Leu Gln Ile 100 105 110 Thr <210> SEQ ID NO 15 <211> LENGTH: 107<212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: REI VL <222> LOCATION: 1..107 <400> SEQUENCE: 15 Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 5 10 15 Asp Arg ValThr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ile Lys Tyr 20 25 30 Leu Asn TrpTyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu AlaSer Asn Leu Gln Ala Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly SerGly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu AspIle Ala Thr Tyr Tyr Cys Gln Gln Tyr Gln Ser Leu Pro Tyr 85 90 95 Thr PheGly Gln Gly Thr Lys Leu Gln Ile Thr 100 105 <210> SEQ ID NO 16 <211>LENGTH: 345 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: Template used to produce HuVH and HuVHA <222> LOCATION:1..345 <223> OTHER INFORMATION: CDR amino acid-encoding codons areindicated by NNN <400> SEQUENCE: 16 cag gtc caa ctg cag gag agc ggt ccaggt ctt gtg aga cct agc cag 48 acc ctg agc ctg acc tgc acc gtg tct ggcnnn nnn nnn nnn nnn nnn 96 nnn nnn nnn tgg gtg aga cag cca cct gga cgaggt ctt gag tgg att 144 gga nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnnnnn nnn nnn nnn 192 nnn nnn nnn nnn nnn nnn nnn nnn gac acc agc aag aaccag ttc agc 240 ctg aga ctc agc agc gtg aca gcc gcc gac acc gcg gtc tatnnn nnn 288 nnn nnn nnn nnn nnn nnn nnn nnn tgg ggc caa ggg tcc ttg gtcacc 336 gtc tcc tca 345 <210> SEQ ID NO 17 <211> LENGTH: 345 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:Template used to produce HuVHS and HuVHK <222> LOCATION: 1..345 <223>OTHER INFORMATION: DNA encoding a humanized heavy chain variable regioncontaining human NEWM FRs and murine CC49 VH CDRs <400> SEQUENCE: 17 caggtc caa ctg cag gag agc ggt cca ggt ctt gtg aga cct agc cag 48 acc ctgagc ctg acc tgc acc gtg tct ggc tac acc ttc act gac cat 96 gca att cactgg gtg aga cag cca cct gga cga ggt ctt gag tgg att 144 gga tat ttt tctccc gga aat gat gat ttt aaa tac aat gag agg ttc 192 aag ggg aga gtg acaatg ctg gca gac acc agc aag aac cag ttc agc 240 ctg aga ctc agc agc gtgaca gcc gcc gac acc gcg gtc tat ttc tgt 288 aca aga tcc ctg aat atg gcctac tgg ggc caa ggg tcc ttg gtc acc 336 gtc tcc tca 345 <210> SEQ ID NO18 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <221> NAME/KEY: Oligonucleotide 10 <222> LOCATION: 1..17<223> OTHER INFORMATION: Oligonucleotide primer for plasmid M13 <400>SEQUENCE: 18 ctaaaacgac ggccagt 17 <210> SEQ ID NO 19 <211> LENGTH: 16<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221>NAME/KEY: Oligonucleotide 11 <222> LOCATION: 1..16 <223> OTHERINFORMATION: Oligonucleotide primer for plasmid M13 <400> SEQUENCE: 19aacagctatg accatg 16 <210> SEQ ID NO 20 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:Oligonucleotide 385 <222> LOCATION: 1..22 <223> OTHER INFORMATION:Oligonucleotide primer for plasmid M13 <400> SEQUENCE: 20 gcgggcctcttcgctattac gc 22 <210> SEQ ID NO 21 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:Oligonucleotide 391 <222> LOCATION: 1..22 <223> OTHER INFORMATION:Oligonucleotide primer for plasmid M13 <400> SEQUENCE: 21 ctctctcagggccaggcggt ga 22 <210> SEQ ID NO 22 <211> LENGTH: 60 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:Oligonucleotide 1a <222> LOCATION: 1..60 <223> OTHER INFORMATION:Mutagenic oligonucleotide for retaining 2 murine FR amino acids in ahumanized VH <400> SEQUENCE: 22 gctgtctcac ccagtgaatt gcatggtcagtgaaggtgta gccagacacg gtgcaggtca 60 <210> SEQ ID NO 23 <211> LENGTH: 60<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221>NAME/KEY: Oligonucleotide 1b <222> LOCATION: 1..60 <223> OTHERINFORMATION: Mutagenic oligonucleotide for retaining 3 murine FR aminoacids in a humanized VH <400> SEQUENCE: 23 gctgtctcac ccagtgaattgcatggtcag tgaaggtgta gccagagcgg gtgcaggtca 60 <210> SEQ ID NO 24 <211>LENGTH: 94 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <221> NAME/KEY: Oligonucleotide 2 <222> LOCATION: 1..94 <223>OTHER INFORMATION: Mutagenic oligonucleotide for retaining 1 murine FRamino acid in a humanized VH <400> SEQUENCE: 24 ctggtgtctg ccagcattgtcactctcccc ttgaacctct cattgtattt aaaatcatca 60 tttccgggag aaaaatatccaatccactca agac 94 <210> SEQ ID NO 25 <211> LENGTH: 60 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:Oligonucleotide 3 <222> LOCATION: 1..60 <223> OTHER INFORMATION:Mutagenic oligonucleotide for retaining 2 murine FR amino acids in ahumanized VH <400> SEQUENCE: 25 ggacccttgg ccccagtagg ccatattcagggatcttgta cagaaataga ccgcggtgtc 60 <210> SEQ ID NO 26 <211> LENGTH: 39<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221>NAME/KEY: Oligonucleotide 21 <222> LOCATION: 1..39 <223> OTHERINFORMATION: Mutagenic oligonucleotide for retaining murine CDR aminoacids in a humanized VL <400> SEQUENCE: 26 gttcttctga ttaccactgtataaaagact tgactggac 39 <210> SEQ ID NO 27 <211> LENGTH: 26 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:Oligonucleotide 22 <222> LOCATION: 1..26 <223> OTHER INFORMATION:Mutagenic oligonucleotide for retaining murine CDR amino acids in ahumanized VL <400> SEQUENCE: 27 cagattccct agcggatgcc cagtag 26 <210>SEQ ID NO 28 <211> LENGTH: 78 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <221> NAME/KEY: Oligonucleotide 23 <222>LOCATION: 1..78 <223> OTHER INFORMATION: Mutagenic oligonucleotide forretaining murine CDR amino acids in a humanized VL <400> SEQUENCE: 28ttctactcac gtgtgatttg cagcttggtc ccttggccga acgtgagggg ataggaatag 60tattgctggc agtagtag 78 <210> SEQ ID NO 29 <211> LENGTH: 23 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: Oligonucleotide24used to produce HuVHS and HuVHK <222> LOCATION: 1..23 <223> OTHERINFORMATION: Mutagenic oligonucleotide for retaining murine CDR aminoacids in a humanized VL <400> SEQUENCE: 29 gctctgggtc atctggatgt cgg 23<210> SEQ ID NO 30 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <221> NAME/KEY: Oligonucleotide 4<222> LOCATION: 1..24 <223> OTHER INFORMATION: Mutagenic oligonucleotidefor retaining a murine serine in FR3 of HuVHS <400> SEQUENCE: 30agacaccagc agcaaccagt tcag 24 <210> SEQ ID NO 31 <211> LENGTH: 24 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221>NAME/KEY: Oligonucleotide 5 <222> LOCATION: 1..24 <223> OTHERINFORMATION: Mutagenic oligonucleotide for retaining a murine serine inFR3 of HuVHS <400> SEQUENCE: 31 gctgaactgg ttgctgctgg tgtc 24 <210> SEQID NO 32 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <221> NAME/KEY: Oligonucleotide 6 <222>LOCATION: 1..24 <223> OTHER INFORMATION: Mutagenic oligonucleotide forretaining a murine lysine in FR3 of HuVHK <400> SEQUENCE: 32 ctggcagacaagagcaagaa ccag 24 <210> SEQ ID NO 33 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:Oligonucleotide 7 <222> LOCATION: 1..24 <223> OTHER INFORMATION:Mutagenic oligonucleotide for retaining a murine lysine in FR3 of HuVHK<400> SEQUENCE: 33 tggttcttgc tcttgtctgc cagc 24

What is claimed is:
 1. An isolated nucleic acid from which may be expressed either: A) a humanized antibody having binding specificity to TAG-72 wherein said humanized antibody comprises: 1) at least one NEWM-grafted humanized heavy chain variable region (VH) having the amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; and 2) at least one REI-grafted humadized light chain variable region (VL) having the amino acid sequence of SEQ ID NO:9; or B) a hunanized antibody fragment having binding specificity to TAG-72, said fragment being a fragment of the humanized antibody of Part A).
 2. An isolated vector comprising a nucleic acid sequence according to claim
 1. 3. The vector according to claim 2 wherein said vector is a bare nucleic acid segment, a carrier-associated nucleic acid segment, a nucleoprotein, a plasmid, a virus, a viroid, or a transposable element.
 4. The nucleic acid according to claim 1 wherein said humanized antibody is the humanized antibody expressed by a cell deposited as ATCC CRL-12209. 